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J M Louarn

Publications and source records attributed to J M Louarn.

At least 37 records · Page 2Linked to original sources
PubMed1987

Inhibition of replication forks exiting the terminus region of the Escherichia coli chromosome occurs at two loci separated by 5 min.

The replication cycle of Escherichia coli strains duplicating their chromosome from the same plasmid origin placed at various locations or of strains having undergone a major inversion event along the origin-to-terminus axis was studied by marker-frequency analysis. It was observed that replication forks are unidirectionally inhibited at two loci of the termination region: counterclockwise-moving forks are inhibited at terminator T1 (28.5 min), and forks moving in the opposite direction are inhibited at terminator T2 (33.5 min). By determining the strand preference of Okazaki fragments that are specific for markers from the T1-T2 interval, it was shown that this interval is replicated in either direction, depending upon the strain analyzed. In addition, we also observed that forks moving in the "unnatural" direction along each oriC-T1 or -T2 arm are very slow, especially in the one-third portion of the chromosome around the terminators. We propose that this phenomenon is a consequence of nucleoid organization, which is proposed to be symmetrical on the two oriC-T1 or -T2 arms and polarized with respect to the direction of replication. We also propose that T1 and T2 are the terminal limits of these two polarized half-nucleoid bodies.

Chromosome Mapping↗
PubMed1986

Suppression of the Escherichia coli dnaA46 mutation by amplification of the groES and groEL genes.

A lambda hybrid phage (lambda Sda1), containing an 8.1 kb EcoRI DNA fragment from the Escherichia coli chromosome, was selected on the basis of its ability to suppress bacterial thermosensitivity caused by the dnaA46 mutation. We have shown that this suppression is due to a recA+-dependent amplification of the 8.1 kb fragment; consistent with this observation, cloning of the 8.1 kb fragment into a high copy number plasmid (pBR325) leads also to suppression of dnaA46. In the suppressed strains growing at high temperature, bidirectional replication starts in or near the oriC region and requires the presence of the DnaA polypeptide. These findings suggest that the overproduction of a gene product(s), encoded by the cloned 8.1 kb fragment, can restore dnaA-dependent initiation of replication at high temperature in the oriC region. Genetic mapping shows that the groES (mopB) and groEL (mopA) genes are located on the 8.1 kb suppressor fragment. Further analysis, including in vitro mutagenesis and subcloning, demonstrates that the amplification of the groES and groEL genes is both necessary and sufficient to suppress the temperature sensitive phenotype of the dnaA46 mutation.

Alleles↗
PubMed1985

Characterization and properties of very large inversions of the E. coli chromosome along the origin-to-terminus axis.

Suppression of a dnaA46 mutation by integration of plasmid R100.1 derivatives in the termination region of chromosome replication in E. coli results in medium dependence, the suppressed bacteria being sensitive to rich medium at 42 degrees C. Derivatives of such bacteria have been selected for growth at 42 degrees C in rich medium and we have analyzed representatives of the most frequently observed type: bacteria displaying, once cured of the suppressor plasmid, both rich-medium sensitivity and temperature sensitivity. We found, in all cases, that the chromosome had undergone a major inversion event between two inverted IS5's. One is located at 29.2 min on the chromosome map and the other at either one of two positions between 69 and 80 min. The consequences of such inversions for cell growth are discussed. Some of them result from the fact that the replication terminator T2 is located, in inverted chromosomes, close to oriC in the orientation which allows its functioning as a terminus (de Massy et al. in press). Our observations allow an estimation of the frequency of inversions arising from recombination between pairs of inverted chromosomal IS, which could be as high as 10(-2) per cell per generation. We also found that inversion reversal occurs frequently after Hfr conjugational transfer of one of the IS5's, in its wild-type location. This led us to propose a new mechanism of recombination, in which the incoming DNA strands serve as guides to favor recombination between the resident sequences.

Chromosome Mapping↗
PubMed1984

Genetic inactivation of topoisomerase I suppresses a defect in initiation of chromosome replication in Escherichia coli.

A strain of Escherichia coli K12 harboring simultaneously the temperature-sensitive dnaA46 mutation and a deletion of the trp-topA-cysB region plates with the same full efficiency at 30 degrees C and 42 degrees C. We have analyzed the possible involvement of the gene coding for topoisomerase I, topA, in this suppression phenomenon. The Ts phenotype was retrieved upon introduction of a plasmid-borne DNA fragment including an active topA gene into this strain, but not upon introduction of the same fragment harboring a topA::Tn1000 insertion. Replication seems to remain DnaA-dependent in the delta (topA) strain, however, since we have been unable to introduce a dnaA::Tn10 allele. We propose either that the dnaA46 gene product is overproduced and compensates for its thermal inactivation, or that initiation at oriC demands less DnaA protein in the absence of topoisomerase I.

Alleles↗
PubMed1984

oriX: a new replication origin in E. coli.

Replication of the chromosome of E. coli at 42 degrees C in an integratively suppressed dnaA mutant (dnaA46 Sin Hfr) occurs predominantly from the origin of replication of the integrated plasmid (oriV). We have carried out a detailed marker frequency analysis on such Hfrs. This analysis indicates that replication at 42 degrees C occurs not only from oriV, but also from an origin, oriX, located in the terminal region of the chromosome close to, but distinct from, the prophage rac (oriJ). In an oxa1 mutant of one of these Hfrs, we have shown that replication proceeds at 42 degrees C from all three origins: oriV, oriX, and oriC. Loss of the integrated plasmid results in a temperature- and rich-medium-sensitive strain that replicates the chromosome from oriC and oriX. Replication from oriX proceeds slowly and bidirectionally. We suggest that oriX may be involved in the coupling between replication and cell division.

Bacterial Proteins↗
PubMed1982

Suppression of Escherichia coli dnaA46 mutations by integration of plasmid R100.1. derivatives: constraints imposed by the replication terminus.

We have studies the phenotypic suppression of a dnaA46 mutation by plasmid integration at preselected chromosomal sites after introducing homologous sequences (Mu prophages) onto both the chromosomes and the suppressive plasmid. The plasmids used were all derived from plasmid R100.1. We found that the conditions required to get viable suppressive integration varied as the plasmid integration site moved from the origin to the terminus of chromosome replication. Two constraints were observed. Both appeared to be linked to the new characteristics acquired by chromosome replication from the integrated plasmid. One constraint was that strains with integrative suppression near the terminus terC were viable only in minimal medium. The rich medium sensitivity of these strains was correlated with a loss of regulation of initiation. The other constraint was a requirement for a specific orientation in certain regions of the chromosome. The two branches defined by normally initiated replication, between oriC and terC, were also symmetrical with respect to these plasmid orientation constraints. In studying the possible reasons for a plasmid orientation constraint, we found that, of the two forks initiated in bidirectional replication from the integrated plasmid, one was capable of moving across the terC region with a higher movability than the other.

Chromosomes, Bacterial↗
PubMed1979

Map position of the replication terminus on the Escherichia coli chromosome.

The directions of replication of several prophages integrated with a known orientation in the vicinity of the terminus (tre) of chromosome replication (trp::Mu, min 27; lambda rev integrated within rac, min 31, man::Mu, min 35), have been established by determining the molecular polarity of Okazaki pieces specific to these prophages. The results obtained strongly suggest that the site tre is located between rac and man, an otherwise genetically silent region.

Chromosome Mapping↗
PubMed1979

Direction of deoxyribonucleic acid transfer and replication in a derivative of plasmid R100-1.

The site of integration and the molecular orientation of a prophage Mu integrated within the resistance transfer factor component of plasmid R100-1 have been determined on the physical map of the plasmid. This allowed us (i) to determine the direction of deoxyribonucleic acid transfer from oriT during conjugation and (ii) to demonstrate the unidirectionality of replication in conditions of exponential growth (by determining the strand preference of Mu-specific Okazaki fragments).

Chromosome Mapping↗
PubMed1978

Map position of the replication origin on the E. coli chromosome.

Strains carrying a dnaA temperature sensitive (t.s.) mutation and a Mu-1 prophage inserted within different genes near the origin of replication have been constructed. For each strain, integratively suppressed Hfrs, named G and D in which the ori region was replicated clockwise and counterclockwise respectively, were isolated. The strand preferences of Mu-1 specific Okazaki fragments were subsequently determined for each t.s. strain and its Hfr derivatives. Their comparison led us to establish the direction of replication of the Mu-1 marker from ori. The site ori was confined to the bglB-C--rbsK-P interval.

Chromosome Mapping↗
PubMed1974

Size distribution and molecular polarity of newly replicated DNA in Escherichia coli.

Newly synthesized DNA, in E. coli lysogenic for the phage lambda, was labeled by short pulses of [(3)H]-thymidine, isolated, and separated on the basis of size by alkaline sucrose density gradient centrifugation. The molecular polarity of this DNA was determined by hybridization with each of the separated strands of lambda DNA. The results show that, in the 3' to 5' direction, replication proceeds by synthesis of short chains that are subsequently joined to long DNA. This is true for both a polA(+) and a polA(-) strain. (The polA locus codes for DNA polymerase I.) In the 5' to 3' direction, replication proceeds continuously, by addition of nucleotides to long DNA, for the polA(+) strain. In the polA(-) strain, however, replication in the 5' to 3' direction is also discontinuous, but the discontinuities are 1-40 times less frequent than in the other direction.

Centrifugation, Density Gradient↗